Anja Mezger

Detection of rolling circle amplified DNA molecules using probe-tagged magnetic nanobeads in a portable AC susceptometer.

Here, the volume-amplified magnetic nanobead detection assay (VAM-NDA) is for the first time applied for detection of rolling circle amplified (RCA) DNA molecules in a portable, commercial AC susceptometer that operates at ambient temperatures and with an analysis time of about 20 min. The performance of the assay is investigated using three different magnetic nanobead sizes: 50, 130 and 250nm. The performance of the assay using the AC susceptometer is compared to the performance achieved using a superconducting quantum interference device (SQUID). It is found that the performance of the assay is comparable in the two setups with a quantitative detection limit of ∼4pM for all bead sizes under study. The findings show that the VAM-NDA holds promise for future wide-spread implementation in commercial AC susceptometer setups thus opening up for the possibility to perform magnetic bead-based DNA detection in point-of-care and outpatient settings.

A General Method for Rapid Determination of Antibiotic Susceptibility and Species in Bacterial Infections

ABSTRACT To ensure correct antibiotic treatment and reduce the unnecessary use of antibiotics, there is an urgent need for new rapid methods for species identification and determination of antibiotic susceptibility in infectious pathogenic bacteria. We have developed a general method for the rapid identification of the bacterial species causing an infection and the determination of their antibiotic susceptibility profiles. An initial short cultivation step in the absence and presence of different antibiotics was combined with sensitive species-specific padlock probe detection of the bacterial target DNA to allow a determination of growth (i.e., resistance) and no growth (i.e., susceptibility). A proof-of-concept was established for urinary tract infections in which we applied the method to determine the antibiotic susceptibility profiles of Escherichia coli for two drugs with 100% accuracy in 3.5 h. The short assay time from sample to readout enables fast appropriate treatment with effective drugs and minimizes the need to prescribe broad-spectrum antibiotics due to unknown resistance profiles of the treated infection.

Sensitive Detection of Spores Using Volume-Amplified Magnetic Nanobeads

A magnetic-nanobead-based, substrate-free method for the sensitive detection of spores in an immunoassay format is presented. The method is shown to detect Bacillus globigii spores, the non-pathoge ...

Scalable DNA-Based Magnetic Nanoparticle Agglutination Assay for Bacterial Detection in Patient Samples

We demonstrate a nanoparticle-based assay for the detection of bacteria causing urinary tract infections in patient samples with a total assay time of 4 h. This time is significantly shorter than the current gold standard, plate culture, which can take several days depending on the pathogen. The assay is based on padlock probe recognition followed by two cycles of rolling circle amplification (RCA) to form DNA coils corresponding to the target bacterial DNA. The readout of the RCA products is based on optomagnetic measurements of the specific agglutination of DNA-bound magnetic nanoparticles (MNPs) using low-cost optoelectronic components from Blu-ray drives. We implement a detection approach, which relies on the monomerization of the RCA products, the use of the monomers to link and agglutinate two populations of MNPs functionalized with universal nontarget specific detection probes and on the introduction of a magnetic incubation scheme. This enables multiplex detection of Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa at clinically relevant concentrations, demonstrating a factor of 30 improvement in sensitivity compared to previous MNP-based detection schemes. Thanks to the universal probes, the same set of functionalized MNPs can be used to read out products from a multitude of RCA targets, making the approach truly scalable for parallel detection of multiple bacteria in a future integrated point of care molecular diagnostics system.

On‐Chip Detection of Rolling Circle Amplified DNA Molecules from Bacillus Globigii Spores and Vibrio Cholerae

For the first time DNA coils formed by rolling circle amplification are quantified on-chip by Brownian relaxation measurements on magnetic nanobeads using a magnetoresistive sensor. No external magnetic fields are required besides the magnetic field arising from the current through the sensor, which makes the setup very compact. Limits of detection down to 500 Bacillus globigii spores and 2 pM of Vibrio cholerae are demonstrated, which are on the same order of magnitude or lower than those achieved previously using a commercial macro-scale AC susceptometer. The chip-based readout is an important step towards the realization of field tests based on rolling circle amplification molecular analyses.

Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ

In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ.

Function and regulation of self-reactive marginal zone B cells in autoimmune arthritis

Polyreactive innate-type B cells account for many B cells expressing self-reactivity in the periphery. Improper regulation of these B cells may be an important factor that underlies autoimmune disease. Here we have explored the influence of self-reactive innate B cells in the development of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis. We show that splenic marginal zone (MZ), but not B-1 B cells exhibit spontaneous IgM reactivity to autologous collagen II in naı¨ve mice. Upon immunization with heterologous collagen II in complete Freund’s adjuvant the collagen-reactive MZ B cells expanded rapidly, while the B-1 B cells showed a modest anti-collagen response. The MZ B cells were easily activated by toll-like receptor (TLR) 4 and 9-ligands in vitro, inducing proliferation and cytokine secretion, implying that dual engagement of the B-cell receptor and TLRs may promote the immune response to self-antigen. Furthermore, collagen-primed MZ B cells showed significant antigen-presenting capacity as reflected by cognate T-cell proliferation in vitro and induction of IgG anti-collagen antibodies in vivo. MZ B cells that were deficient in complement receptors 1 and 2 demonstrated increased proliferation and cytokine production, while Fcγ receptor IIb deficiency of the cells lead to increased cytokine production and antigen presentation. In conclusion, our data highlight self-reactive MZ B cells as initiators of the autoimmune response in CIA, where complement and Fc receptors are relevant in controlling the self-reactivity in the cells.Cellular & Molecular Immunology advance online publication, 11 May 2015; doi:10.1038/cmi.2015.37

Detection of Rotavirus Using Padlock Probes and Rolling Circle Amplification

Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus.

Multiplexed protein profiling by sequential affinity capture

Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off‐target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi‐automated sequential capture assay. This novel bead‐based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read‐out by a secondary capture bead array. We demonstrate in a proof‐of‐concept setting that target detection via two sequential affinity interactions reduced off‐target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA‐based signal amplification, and demonstrate the applicability of the dual capture bead‐based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond.

Highly specific DNA detection employing ligation on suspension bead array readout

We show for the first time that monomerized rolling circle amplification (RCA) products can be directly detected with the Luminex suspension bead array readout without the need of PCR amplification. Furthermore, using monomerized RCA products to guide ligation of the detection oligonucleotide (DO) to barcode sequences on the magnetic Luminex beads, combined with efficient washing and increased measurement temperature, yields a higher signal to noise ratio. As a proof-of-principle, we demonstrate detection of pathogenic DNA sequences with high reproducibility, sensitivity and a dynamic range over four orders of magnitude. Using padlock probes in combination with bead suspension arrays opens up the possibility for highly multiplexed DNA targeting and readout.